1. Field of the Invention
The present invention relates to a method for enhancing the production of polyhydroxyalkanoic acid (PHA), and more particularly, to a method for enhancing the production of polyhydroxyalkanoic acid (PHA) from microorganism strains by genetic manipulation of a wild-type microorganism strain.
2. Description of the Related Art
A variety of microorganisms are known to produce intracellular energy and carbon storage compounds known as polyhydroxyalkanoates (PHAs). PHAs have good thermoplastic properties, biodegradability, biocompatibility and other excellent traits which have attracted considerable academic and industrial interest. According to their side chain lengths, PHAs are divided into short-chain-length (SCL-) PHAs and medium-chain-length (MCL-) PHAs. The metabolic pathways used for bacterial MCL-PHA biosynthesis have been well known as two major routes found in Pseudomonas: (1) de novo fatty acid biosynthesis pathway, which produces (R)-3-hydroxyacyl-CoA precursors from non-related carbon sources, such as glucose and gluconate; and (2) fatty acid degradation by β-oxidation, which is the main metabolic route of fatty acids.
Many researchers produced PHAs using different types of techniques such as PHA synthesis-related gene insertion, a combination of different precursor carbon sources, multistep cultures, and the pathway routing by inhibitors. Although genes and their products directly related to MCL-PHA biosynthesis have been studied, little is known about the roles of other genes and gene products that may be indirectly involved in the PHA synthesis.
Extracellular polymeric substances (EPS) can be produced by various bacteria and perform important functions for the secreting organisms, including cell attachment or locomotion, protection from desiccation, resistance to toxins, and enhancement of their ability to sequester nutrients. According to its relative proximity to the cell surface, the EPS occur in two forms: (1) as capsular EPS, which is tightly linked to the cell surface via a covalent or noncovalent association; and (2) as slime EPS, which is loosely bound to the cell surface. The EPS is mainly composed of polysaccharides, proteins, lipids, a small amount of hexane, and other biopolymers. The composition and location of the PHA depend on several metabolic processes such as changes in growth phase, cell breakage due to cell death, active secretion, release of cell surface macromolecules (outer membrane proteins and lipopolysaccharides), and interaction with the environment. EPS biosynthesis and composition vary from one bacterial species to another and have been shown to be controlled by several environmental factors such as growth phase, growth media, temperature, limitation of oxygen and nitrogen, and cation deficiency. In recent years, interest in the exploitation of valuable EPS has been increasing for various applications in the food and pharmaceutical industries, heavy metal removal, and wastewater treatment, etc. EPS is also considered an abundant source of structurally diverse polysaccharides, some of which may possess unique properties for special applications. Polysaccharide biosynthesis requires an enzyme related to the synthesis of sugar nucleotide precursors as polysaccharide building blocks as well as a specific polysaccharide synthetase. Uridine triphosphate (UDP) glucose pyrophosphorylase (GalU) catalyzes the reversible formation of UDP-glucose and inorganic pyrophosphate (PPi) from UTP and glucose 1-phosphate. UDP-glucose not only functions as a precursor for polysaccharide biosynthesis but is also involved in the biosynthesis of several cell wall components. UDP-glucose is the substrate for the synthesis of UDP-glucuronic acid, and is also required for the interconversion of galactose and glucose by the Leloir pathway.
The inventors of the present invention have reported in a previous study that Pseudomonas fluorescens BM07 (hereinafter referred to as P. fluorescen BM07) secreted large amounts of exobiopolymer (EBP) when grown on fructose at 10° C. and played an important role in the bioremediation of heavy metals, especially in the cold season. The main components of the cold-induced EBP in BM07 are water-insoluble hydrophobic polypeptide(s) (up to 85%) and saccharides (8%). Carbohydrate analyses revealed glucose, glucosamine, and galactosamine as major components of the sugar units in the EBP. The isolated EBP exhibited an endothermic transition with an enthalpy of 84 J/g at 192° C. as well as a sharp X-ray diffraction pattern, suggesting a probable uniquely structured organization around cells.
The inventors of the present invention propose the production and characterization of P. fluorescens BM07 transposon mutants, which are disrupted in EBP formation but increase PHA accumulation compared with the wild type.